Transduction of Human Embryonic Stem Cells by Foamy Virus Vectors Helen Gharwan, Roli K Hirata, Peirong Wang, Robert E Richard, Linlin Wang, Erik Olson, James Allen, Carol B Ware, David W Russell  Molecular Therapy  Volume 15, Issue 10, Pages 1827-1833 (October 2007) DOI: 10.1038/sj.mt.6300244 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Transduction by foamy virus (FV) vector ΔΦPP. (a) Map of the proviral form of the FV vector ΔΦPP. The positions of the Southern blot probe and the EcoNI, EcoRI, and EcoRV restriction sites are shown. (b) H1 human embryonic stem cells were infected in the presence of mouse embryonic fibroblast feeder layers with the indicated amounts of ΔΦPP vector stock, and the number of puromycin-resistant clones observed 14 days after selection is shown. LTR, long terminal repeat; PGK, phosphoglycerate kinase. Molecular Therapy 2007 15, 1827-1833DOI: (10.1038/sj.mt.6300244) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Transduction by foamy virus (FV) vector ΔΦPF. (a) Map of the proviral form of the FV vector ΔΦPF. The positions of the Southern blot probe and the EcoNI, EcoRI, and EcoRV restriction sites are shown. (b) Phase-contrast and fluorescence images of a colony of H1 human embryonic stem cells (hESCs) transduced by ΔΦPF and exhibiting homogeneous green fluorescent protein (GFP) expression in all cells at passage 10 after transduction. (c) Flow cytometry of transduced and untransduced H1 hESCs obtained 8 days after infection with ΔΦPF at a multiplicity of infection (MOI) of 5 in the absence of mouse embryonic fibroblast (MEF) feeder layers. The red fluorescence channel serves as a control for auto-fluorescence. (d) H1 hESCs were infected in the absence of MEF feeder layers with ΔΦPF at the indicated MOIs, and the percentage of GFP+ cells is shown. The original flow data for the MOI of 5 infection is shown in (c). LTR, long terminal repeat; PGK, phosphoglycerate kinase. Molecular Therapy 2007 15, 1827-1833DOI: (10.1038/sj.mt.6300244) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Southern blot analysis of transduced embryonic stem cells (ESCs). (a) Genomic DNA samples from puromycin-resistant colonies obtained after transduction of H1, BG03, and rhES366.4 ESCs with foamy virus (FV) vector ΔΦPP at a multiplicity of infection of 1 were digested with EcoRI and probed with an FV vector probe. Sample numbers indicate cultures derived from different colonies. H1 control cells were not transduced. (b) As in (a), but the transduced colonies were passaged for a prolonged period. (c) As in (b), but digested with EcoNI. (d) Southern blot analysis of H1 cells transduced by ΔΦPF and passaged by repeated dissection of green fluorescent protein–positive cells, digested with the indicated enzymes, and probed with the FV probe. The positions of size standards are indicated to the left of each panel. The locations of restriction sites and the probe are shown in Figures 1 and 2.kb, kilobases. Molecular Therapy 2007 15, 1827-1833DOI: (10.1038/sj.mt.6300244) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Gene expression in undifferentiated, transduced human embryonic stem cells (hESCs). (a) Reverse transcriptase polymerase chain reaction (PCR) analysis of four clones of ΔΦPP-transduced H1 hESCs were analyzed at passage 12 after transduction (lanes 2–5). ΔΦPF-transduced H1 hESCs were analyzed at passage 20 after transduction (lane 6). The differentiated negative controls were normal human fibroblasts (lane 7). Additional controls included samples processed without reverse transcriptase (lane 8), or without complementary DNA (lane 9). The primers shown in Table 2 were used to amplify sequences of the indicated genes. A 100-base-pair (bp) ladder was used to determine the sizes of the PCR products. The predicted sizes are indicated. (b) Expression of cell surface markers on transduced hESCs. The left panels show individual colonies of ΔΦPF-transduced H1 cells expressing green fluorescent protein (GFP). The right panels show the same colonies stained with Texas Red for surface antigens characteristic of undifferentiated pluripotent hESCs (Oct-4, TRA-1-60 and TRA-1-81, SSEA-3, and SSEA-4) or stained for SSEA-1 as a negative control. Molecular Therapy 2007 15, 1827-1833DOI: (10.1038/sj.mt.6300244) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Reverse transcriptase polymerase chain reaction (PCR) analysis of differentiated, transduced human embryonic stem cells. ΔΦPF-transduced and untransduced H1 cells were analyzed with or without reverse transcription as indicated for expression of α-fetoprotein (AFP; endoderm), renin (mesoderm), and neurofilament heavy chain (NFH; ectoderm). As a negative control samples without complementary DNA were processed (H2O control). A 100-base-pair (bp) ladder served to determine the sizes of the PCR products, with the expected sizes shown on the right. Molecular Therapy 2007 15, 1827-1833DOI: (10.1038/sj.mt.6300244) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 Teratoma formation by green fluorescent protein (GFP)+–transduced cells. Representative gross specimen of a teratoma examined under (a) fluorescent or (b) bright light. Frozen histological section of a teratoma stained with hematoxylin and eosin and examined under (c) low and (d, e) high power. (f–h) Sections from teratomas stained with hematoxylin and eosin (H&E), 4,6-diaminidino-2-phenylindole (DAPI), an antibody against GFP, or antibodies against α-smooth muscleactin (ASM), mouse anti-microtubule-associated protein-2 (MAP-2), or α-fetoprotein (AFP) as indicated. The first four panels of each row are fluorescent images from the same section, and the H&E panels in the same row are from adjacent sections. Scale bars represent (a, b) 6 mm, (c) 370 μm, or (d–h) 60 μm. Molecular Therapy 2007 15, 1827-1833DOI: (10.1038/sj.mt.6300244) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions